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The Agilent miRNAMicroarray System

Udo Schimmel24.Juli 2007

A New Microarray-based Tool for

Profiling Human miRNAs

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Choose from a comprehensive set of genomic applications

DNA RNA

Splice VariantsChIP

• Elucidate the role that protein-DNA

interactions play in transcription,

replication, modification and

repair

• Perform global interrogations of

the transcriptomeand identify

alternative splice forms to uncover

the role gene variants play in

drug response and disease

GX

• Explore gene transcription on a

genome-wide basis across a

variety of model systems

mRNA

aCGH

• Conduct high-resolution,

genome-wide profiling of

DNA copy number changes

associated with cancer and

other genetic diseases

Copy number

CH3

� Discover and monitor

epigenetic modifications

known to play a fundamental role

in many cellular processes

Methylation Transcription Factors

mRNA isoforms

miRNA

• Profile microRNAsand explore the role

they play in gene regulation

RNA interference

From one-dimensional

with multiple applications …

to multi-dimensional …

Established applications New applications

miRNA

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miRNA Scientific Background and Importance

� Definition: 19-30 nucleotide long single-stranded RNAs that post-transcriptionally regulate gene expression

� Key regulators of gene expression, development, proliferation, differentiation, and apoptosis

� May regulate >30% of human genes

� Current Sanger miRBASE Release 9.1 has 4361 entries, 474 identified in humans

� Projected $100M to be awarded by the NIH for miRNA-related research in 2008*

� Discovery of new miRNAs is ongoing …

*Source: NIH CRISP database at http://crisp.cit.nih.gov/

Cancer Genomics: Small RNAs with big impacts from Nature 435: 745-746 (9 June 2005)

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AAAAAAAAAA5’

Mature miRNA sequences are embedded in one sides of the ~30bp stems of the hairpins

• Two types of miRNA genes:

• Independent transcription units transcribed by RNA Polymerase II– Primary transcripts are capped and polyadenylated– Transcripts may contain more than 1 miRNA– Two exceptions so far:

• Mouse herpesvirus 68 miRNA genes are Pol III transcribed• Adenovirus VA1 genes Pol III transcribed

• Found in introns of mRNA genes (also Pol II transcripts)– Precursors are then processed after intron removal

• These long, primary precursor RNAs are called pri-miRNAs.

miRNAs – How Are They Transcribed?

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miRNAs – How Are They Processed?

AAAAAAAAAA5’

• Pri-miRNA

• Pre-miRNAs

• Mature miRNAs

Drosha (endonuclease)DGCR8 (dsRNA-binding protein)

Dicer (endonuclease) (same enzyme used in RNAi)TRBP (dsRNA-binding protein)

Dicer cuts out mature miRNAfrom stem of stem loop

Other strand is also cut out –This strand is either degraded,or can also be used as an miRNA

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microRNA Function in Animals

miRNA

Binds RISC Proteins

miRNA bound to RISC base-pairs to mRNA

Translation blocked!

Ribosome

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Growth of miRNA Publications

Source: PubMed search with terms “miRNA” or “microRNA”

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Certain microRNAs (miRNA) findings

• 19-30 nts single-stranded RNAs

• Key regulators of cell development

• Control protein production

• Found in 32 animals, 9 plants, & 8 viruses ( total > 4400)

• >470 identified in humans

• Expressed from intergenic regions and introns

• May regulate >>30% of human genes

• Tissue-specific expression patterns

• Expressed in large dynamic range (>4 orders of magnitude)

• Different cancers have distinct miRNA expressions

• Diagnostic potential being explored

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miRNAs – How many Genes do they Control?

• Cell 126: 1203 (2006) K.Miranda et al.– Using computational techniques, suggest that most human mRNAs have at least

one miRNA binding site in 3’ UTR

– Also predict binding sites in coding regions and 5’ UTRs of many genes

– Using another computational technique, suggest there may be 1000s of miRNAs in humans

• Each miRNA can have 10s-1000s of targets– Suggests vast, complex regulatory networks

• Are most human genes regulated by miRNA?

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Certain miRNA findings in Cancer

� Cancer cells generally have lower levels of miRNAs than normal cells

� Depending on the genes they regulate, miRNAs can function as either an oncogene or tumor suppressor

� miRNA profiles can be used to classify human tumors (Lu et al., Nature 435, 834 (2005)- Profiling of 217 miRNAs from 334 tumors yielded highly accurate classification

- Classifications reflects developmental lineages and tissue of origin

� Alterations of miRNA expression have been observed in a number of cancers- Colorectal, pituitary, lung, breast, B-cell lymphoma, glioblastoma, others

- In several cancers, specific miRNA signatures are associated with prognosis

� Using CGH, shown that miRNA loci have a high frequency of genomic alterations in human cancers (Zhang et al, PNAS 103, 9136 (2006)

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1. Small size

2. High sequence homology

3. Presence of precursors

4. Expressed with large dynamic range

5. Growing database

Challenges in miRNA Profiling

miRNA Sequence #NT

hsa-let-7a ugagguaguagguuguauaguu 22

hsa-let-7b ugagguaguagguugugugguu 22

hsa-let-7c ugagguaguagguuguaugguu 22

hsa-let-7d agagguaguagguugcauagu 21

hsa-let-7e ugagguaggagguuguauagu 21

hsa-let-7f ugagguaguagauuguauaguu 22

hsa-let-7g ugagguaguaguuuguacagu 21

hsa-let-7i ugagguaguaguuugugcugu 21

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Direct and Sensitive miRNA Profiling

Back to Applications

Platform

Hui Wang, PhD

Senior Scientist

Agilent Technologies

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Isolating microRNA

When isolating RNA for microRNA purification, do not use a standard column based cleanup. These columns eliminate small RNAs from the sample.

1. Ambion mirVana™ miRNA Kit. Designed for isolation of smallRNA. Overnight EtOH precipitation (>90% yield) or flashPage (>70% yield) fractionation can be used.

2. Trizol isolation, but instead of EtOH precipitation the aqueous phase is passed through a Millipore YM-100 column, and the flow-through is collected. The flow-through is passed through a YM-3column, and this time the remain on the filter is collected.The collected material is EtOH precipitated

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Detection and Analysis of Small RNA

Agilent Technologies

Lab-on-a-Chip solutions

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Small RNA Assay versus existing RNA Assay

Low

er

Mark

er

tRNA

18s rRNA 28s rRNA

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Low

er

Mark

er

tRNA

18s rRNA 28s rRNA

Small RNA Assay versus existing RNA Assay

New Small RNA Assay

RNA 6000 Assay

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The new Assay as a tool for:

miR

NA

tRN

A rRN

A

LM

Intact Total RNA sample

Verification, comparison and optimization for the small RNA region:

• High sensitivity to detect low abundant fragments

• High resolution for ss oligos, miRNA, pre-, t-, 5S-RNA’s

• compatible with Total RNA samples or purified small RNAs.

• Semi-quantitative for single stranded RNA.

• Denaturing

• Analysis up to 150nt

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Small RNA Assay

Same product structure as with existing RNA nano or pico:

RNA Ladder shows high resolution optimized for the 20nt range.

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Calculated Concentration [pg/µl] -

RegionName: miRNA

Avg

Siz

ee

Semi quantitative Smear Analysis

y = 0.8077x + 12.098

R2 = 0.9924

19.2

219.2

419.2

619.2

819.2

1019.2

1219.2

1419.2

1619.2

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Nominal Concentration [pg/µl] -

RegionName: miRNA

Reg

ion

Co

ncen

tra

tio

n• Mixture containing 80 synthetic RNA oligos, from Human miRNA

Sanger database. Size range of 18-23 nucleotides, analyzed in 5 different concentrations

Typical electropherogram: sequence differences account for the smeared size range covered

Excellent linearity

Calculated size Average for the miRNA region is in accordance with expected values.

miR

NA

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Monitoring purification of microRNA samples

totalRNA pre-purified with a commercial smallRNA kit (cut-off 200nt) further enriched using preparative polyacrylamide gel (cut-off 40nt)

Residual tRNA

tRNA

MicroRNA

miRNA to tRNA ratio was increased by a factor of ~20

MicroRNA

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HEK 293 Cells: direct miRNA extraction

using the Ambion „mirVana“ Kit and RNA 6000nano

Small RNA depleted fraction Small RNA enriched fraction

Existing RNA 6000 nano kit(no Lower Marker used)

Monitoring purification of microRNA samples

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FlashPAGE Purification of miRNA

miRNA Peak

Pre-purified

small RNA (0-150 nt)

“Flash

PAGE” purified miRNA

Monitoring purification of microRNA samples

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Agilent miRNA Profiling Method

Simple Labeling Protocol

• Enzymatic direct end-labeling

• High yield & low sequence bias

• Low input total RNA

• No amplification

• No RNA size fractionation

• 1 Cy/target molecule

• 1 color hybridization

Probe Design

• Specific to labeling method

• Applicable to human and other

species

• Sequence & size specific

• Empirically Tm-balanced

• Can easily incorporate new

miRNAs

• Probes for all human miRNAs in

Sanger Database Release 9.1

(Feb 2007)

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Simple Work Flow

Total RNA (100ng)Total RNA (100ng)Total RNA (100ng)Total RNA (100ng)

DephosphorylatedDephosphorylatedDephosphorylatedDephosphorylated RNARNARNARNA

miRNA ProfilemiRNA ProfilemiRNA ProfilemiRNA Profile

Phosphatase Treatment, incubate 30 min, 37oC

*The sample can

be stored frozen

at -20oC if

necessary

Add DMSO

Heat, IceAssemble Labeling Reaction, incubate 16oC 2hr

Labeled RNALabeled RNALabeled RNALabeled RNA

*

Desalt with Spin Column

Assemble Hybridization Mixture

Desalted Labeled RNADesalted Labeled RNADesalted Labeled RNADesalted Labeled RNA

Heat, Ice

Hybridize 20 hours, 55oC, 20RPM

Wash, Scan

*

*

Entire labeling protocol can be easily completed within one afternoon, starting with total RNA

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Agilent Synthesis Technology

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Advantages• Free content selection• High sensitivity• Cost efficient

In situ Synthesis of 60-mer Oligonucleotides

Agilent InkJet Printing Technology

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Agilent ‘8-pack’ DNA Microarrays

Array Design

• 8 identical microarrays per slide

• 15,000-features per microarray

• Generally: 2-4 probe sequences/ miRNA

• Multiple features per probe sequence

• Robust total gene signal measurement

Signal of each miRNA are reported as the sum of all

signals from the corresponding features

Oligo Mix (50amol/miRNA)) Placenta (100ng) Thymus (100ng) Liver (100ng)

Heart (100ng) Brain (100ng) Testes (100ng) Breast (100ng)

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Probe Design Strategy

Start design with full-length miRNA-

probe sequence, attached to a stilt

sequence.

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Probe Design Strategy

Utilize the C incorporated during

labeling for additional G-C base

pair on 3’end of miRNA to increase stability

Start design with full-length miRNA-

probe sequence, attached to a stilt

sequence.

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Probe Design Strategy

Utilize the C incorporated during

labeling for additional G-C base

pair on 3’end of miRNA to increase stability

Sequentially shorten

target-probe base pairing from 5’ end of miRNA during

preliminary Tm balancing by

calculation.

Start design with full-length miRNA-

probe sequence, attached to a stilt

sequence.

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Probe Design Strategy

Utilize the C incorporated during

labeling for additional G-C base

pair on 3’end of miRNA to increase stability

Sequentially shorten

target-probe base pairing from 5’ end of miRNA during

preliminary Tm balancing by

calculation.

Incorporate hairpin

structure on probes to increase size specificity and

possibly stability.

Final Step: Select Tm-balanced probes for each miRNA empirically using microarray data.

Start design with full-length miRNA-

probe sequence, attached to a stilt

sequence.

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Optimized Labeling Yield

Six different miRNA sequences tested for DMSO optimization

Pre-heated RNA

No pre-heat

treatment

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•220 of individual reactions shown from 47 different sequences

•Ligation of 47 sequences were repeated 1-10 times

Minimal Sequence Bias

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Array Design

• The microarray represents 470 human miRNAs and 64 human viral miRNAs

• 19 miRNAs are represented by 40 features and the rest by 20

• 246 miRNAs have 2 probe sequences (10 features per sequence)

• 192 have 4 probe sequences (5 features per sequence)

• 77 have 3 probe sequences (1 sequence with 6 features and 2 sequences with 7 features

• Probes have been empirically Tm matched when possible (when the miRNAs they represent were detected in any of the tissue samplestested)

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TotalGeneSignal Calculation

• Hybridization reaches almost equilibrium thanks to the array design, the labeling scheme and the hybridization conditions.

• The sum of the signals across all the features corresponding to a given miRNA (TotalGeneSignal) is very stable, independent of the number of probes representing that miRNA. (not so for average signal)

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TotalGeneSignal Calculation cont.

• To provide robust measurements and to ensure compatibility across future array designs with different numbers of probe replicates we calculate TotalGeneSignal rather than average signal per feature:

– Outliers are identified and rejected

– The TotalProbeSignal is calculated as the sum of the signals of all the pixels of all the features for that Probe.

• Calculate the average of counts per pixel per sequence

• Multiply the number above by the total number of pixels per feature (fixed) and features per sequence This gives us theTotalProbeSignal.

– Add the Signal Values for the different probes per miRNA and we obtain the TotalGeneSignal value

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Purified Small RNA (Cy3)

To

tal R

NA

(C

y3

)

Total RNA profiles were compared with small RNA from FlashPAGE purified samples (heart, liver, thymus, brain, and breast total RNAs from Ambion). Typical correlation shown above.

Purified Small RNAs (60ng from 570ng total RNA) vs. Total RNA (120ng from Placenta)

Direct Measurement of miRNA from Total RNA

Data shown are background-subtracted signals with no filtering or normalization.

Each miRNA has

signals from multiple probes.

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miRNA Profiles Using 100ng Total RNA

Placenta Placenta

Pla

cen

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Bra

in

Reproducibility Differential Expression

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Specificity to Distinguish Homologous miRNAs

miRNAs

hsa-let-7a

hsa-let-7bhsa-let-7c

hsa-let-7d

hsa-let-7ehsa-let-7f

hsa-let-7g

hsa-let-7i

UGAGGUAGUAGGUUGUAUAGUU

UGAGGUAGUAGGUUGUGUGGUU

UGAGGUAGUAGGUUGUAUGGUU

AGAGGUAGUAGGUUGCAUAGU

UGAGGUAGGAGGUUGUAUAGU

UGAGGUAGUAGAUUGUAUAGUU

UGAGGUAGUAGUUUGUACAGU

UGAGGUAGUAGUUUGUGCUGU

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2222

21

2122

2121

miRNA Sequence Length (nts)

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85 - 100%

70 - 84%

55 - 69%

40 - 54%25 - 39%

10 - 24%

5 - 9%

0 - 4% Grey Number0

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Oligo Mix Hyb Titration

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RNA Amount (zmol)

70 Equal-molar synthetic miRNAs were labeled and hybridized at 0.01 amol to 1 fmol/miRNA per microarray.

• Synthetic miRNAs were selected for typical and atypical sequence representations.

• Lowest & highest calculated Tms are represented.

• Most miRNAs are detectable at 0.1 amol.

• 10 zmol ~ 6000 molecules

105 Linear Dynamic Range

1 fmol = 1000 amol1 amol = 1000 zmol

To

tal

Sig

nal

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Correlation between replicate arrays

X-axis: Cogenics (Default Interpretation) : Brain A...

Y-axis: Cogenics (Default Interpretation) : Brain A...

Colored by: Cogenics, Default Interpretation (Brai...

Gene List: all genes (532)

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Brain Amount 100.0 , Number 2 (raw)

X-axis: Cogenics (Default Interpretation) : Brain A...

Y-axis: Cogenics (Default Interpretation) : Brain A...

Colored by: Cogenics, Default Interpretation (Brai...

Gene List: all genes (532)

0.01 0.1 1 10 100 1000 1e4 1e5 1e6

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Brain Amount 100.0 , Number 4 (raw)

Bra

in 1

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Bra

in 3

Brain 4

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Distinct Profiles from Different Tissue RNAs

High Signal

Low Signal

Color Key

High Signal

Low Signal

Color Key

hsa-miR-193ahsa-miR-210hsa-miR-490

hsa-miR-30e-3phsa-miR-30a-3p

hsa-miR-186hsa-miR-197hsa-miR-208

hsa-miR-126*hsa-miR-189

hsa-miR-302ahsa-miR-302dhsa-miR-302b

hsa-miR-302a*hsa-miR-367

hsa-miR-122ahsa-miR-148a

hsa-miR-192hsa-miR-194hsa-miR-215

hsa-miR-146bhsa-miR-147hsa-miR-384

hsa-miR-181ahsa-miR-142-3p

hsa-miR-16hsa-miR-15ahsa-miR-19ahsa-miR-205hsa-miR-20bhsa-miR-19b

hsa-miR-17-5phsa-miR-150

hsa-miR-106bhsa-miR-200b

Bra

in

Bre

ast

Heart

Liv

er

Th

ym

us

Pla

cen

ta

Sk. M

uscle

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miR-34a

miR-96 miR-139

miR-141 miR-150

miR-206 miR-218

miR-335 miR-424

miR-24

miRNA levels in each tissue are expressed as the fraction of the expression level in the tissue where that miRNA is most abundant.

qRT-PCR reactions and microarray hybridizations were each repeated four times for each miRNA.

Microarray DataqPCR Data

Quantitative PCR correlation

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We have shown for the first time that let-7 expression is frequently reduced in lung cancers

and that alterations in the miRNA expression may

have a prognostic impact on the survival of surgically treated lung cancer patients. Agilent miRNA arrays

give us the comprehensive miRNA expression profile

with excellent performance on sensitivity and accuracy.I expect that the studies of Agilent miRNA array

may ultimately provide a foundation for a

new paradigm of the involvement of miRNA in human oncogenesis. Dr. Takashi Takahashi

Professor of OncologyMolecular Carcinogenesis

Nagoya University

Response from Early Access Customer

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Agilent miRNA Platform Highlights

� Low sample input - 100 ng of total RNA

� No small RNA isolation required

� Simple one-tube protocol – results in < 2 days

� High sequence and size specificity – mature miRNAs

� High sensitivity – Detection limit <0.1 αmol

� Broad linear dynamic range (>4 logs)

� Replicate probes per array (20 per miRNA)

� One-color analysis

Enabled by Agilent miRNA Probe Design and Direct Labeling Methodology

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Summary

• Efficient labeling method

• Low sample input

• Simple workflow

• Sequence & size specificity

• Large linear dynamic range

• Robust total RNA profiling

• qRT-PCR correlation

• Application to FFPE samples

• Flexible platform for new sequences

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ACKNOWLEDGMENTS

HUI WANG, ROBERT A. ACH, and BO CURRY

Agilent Technologies, Inc., Agilent Laboratories, Santa Clara,California 95051, USA

Dr. Michael Bittner,Translational GenomicsResearch Institute, for theFFPE samples,

Dr. Jeff Sampson for early support and for the probehairpin sequencedesign,

Dr. Nick Sampas for Tmcalculation software,

Dr. Brian Peter

Dr. Steve Laderman

Dr. Laurakay Bruhn

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Questions please..

•Thank You!!Thank You!!Thank You!!Thank You!!